Osteopontin (hereinafter referred to as “OPN”) is an acidic calcium-binding glycoprotein abundantly found in the bone, and in the case of humans, it is known that at least three isoforms can occur due to differences in mRNA splicing: osteopontin-a (hereinafter referred to as “OPN-a”), osteopontin-b (hereinafter referred to as “OPN-b”) and osteopontin-c (hereinafter referred to as “OPN-c”) (non-patent document 1). In particular, the precursor of OPN-a has the amino acid sequence shown by SEQ ID NO:23 in the sequence listing given below, and is considered to undergo signal peptide cleavage upon secretion to form the mature form OPN-a of I17-N314. The mature form of OPN is cleaved by thrombin in vivo on the C-terminal side of the 168th (in the case of OPN-a) arginine residue, resulting in an N-terminal fragment and a C-terminal fragment.
The above-described OPN is responsible for a wide variety of physiologically and pathologically important functions, and has functions, for example, cell adhesion, cell migration, tumorigenesis, immune responses, inhibition of complement-mediated cytolysis, and the like. These diverse functions are mediated by a wide variety of cell surface receptors. OPN has the RGD sequence therein (for example, for OPN-a, 159th to 161st residues); integrins that recognize this RGD sequence, such as αVβ3, αVβ1 and αVβ5, are major receptors of OPN, of which αVβ3, αVβ1 and αVβ5 integrins mediate cell adhesion in vascular smooth muscle cells; furthermore, αVβ3 is associated with the migration of macrophages, lymphocytes, endothelial cells, smooth muscle cells and the like.
Furthermore, research that has been conducted to date has also demonstrated that OPN binds to α9β1, α4β1 and α9β1 integrins via the SVVYGLR sequence (SEQ ID NO:10), and a difference in binding mode has been found in that α4β1 binds to both OPN not cleaved by thrombin (non-cleaved type OPN) and an N-terminal fragment cleaved by thrombin (cleaved type OPN), whereas α9β1 binds only to thrombin-cleaved type OPN (non-patent documents 2 to 4). These α9 and α4 and β1 and β7 integrin subunits are highly similar to each other in terms of amino acid sequence. α4β1 and α4β7 integrins are found mainly in lymphocytes and monocytes but expressed at very low levels in neutrophils. On the other hand, α9β1 is highly expressed selectively in neutrophils, and is responsible for the essential functions for neutrophil migration via VCAM-1, Tenascin-C and the like. α9β1 is widely expressed in myocytes, epithelial cells, hepatocytes and the like. Hence, the cytoplasmic domains of the integrin subunits α4 and α9 are considered to be involved in various inflammatory reactions by cooperatively promoting the migration and aggregation of leukocytes to inflammation sites via respective slightly different intracellular signal transduction pathways to enhance the infiltrating activities thereof.
As described above, because a wide variety of integrins promote the migration of leukocytes and are involved in inflammatory reactions, drugs that inhibit these integrin activities are thought to have the potential for serving as anti-inflammatory agents. For example, integrin αVβ3 is expressed in osteoclasts, vascular endothelial cells, smooth muscle cells and the like; because inhibiting the binding of αVβ3 integrin and various binding ligands thereof is expected to have joint destruction suppressive action in, for example, joints, development of anti-αVβ3 antibody is actually ongoing.
However, because receptors belonging to the integrin family are universally expressed in a broad range of tissues and responsible for the essential functions for the maintenance of biological activities, use of an antibody against integrin in the treatment of rheumatoid arthritis or osteoarthritis can cause similar inhibition in other sites, and the onset of adverse reactions is of concern.
From this viewpoint, attempts have been made to date to clarify the etiology of rheumatoid arthritis, osteoarthritis and the like, and to provide a better therapeutic method.
For example, in WO02/081522 (patent document 1), it was found that in rheumatism patients and osteoarthritis patients, the OPN concentration of articular cavity fluid had high values, and in rheumatism patients, the ratio of thrombin-cleaved type N-terminal fragment to the total OPN increased, and it was confirmed that OPN was profoundly associated with the onset of these diseases. In patent document 1, antibodies that discretely recognize the N-terminal fragment and C-terminal fragment resulting from cleavage of OPN with thrombin, respectively, were generated, and a study using them showed that in rheumatoid arthritis patients, the thrombin-cleaved N-terminal fragment, in particular, exhibited high concentrations in the articular cavity. In this N-terminal fragment, the RGD sequence and the SVVYGLR sequence (SEQ ID NO:10), both recognized by human type integrins, coexist; an antibody that simultaneously blocks these two sequences has been confirmed to be widely inhibit the binding of OPN and integrin, and to be effective in the treatment of rheumatoid arthritis, osteoarthritis and the like.
Specifically, in patent document 1, an antibody that inhibits the binding between the RGD sequence of human OPN and integrin and the binding between the SVVYGLR sequence of human OPN (SEQ ID NO:10) and integrin was generated, and its effect was confirmed by experiments on cell adhesion, cell migration and the like. Furthermore, an antibody against a synthetic peptide corresponding to the internal sequence of mouse OPN was acquired, and its effect as a therapeutic drug was confirmed using a mouse pathologic model of arthritis.
Hence, since mouse OPN has the RGD sequence and the SLAYGLR sequence (SEQ ID NO:12), both recognized by mouse integrin, at positions on amino acid sequence homologous to those of human OPN, the M5 antibody was acquired as an antibody that simultaneously blocks these sequences. It was confirmed that the binding of this M5 antibody to mouse OPN and the thrombin-digested product thereof was inhibited by the GRGDSP peptide, which comprises the RGD sequence, and that this M5 antibody inhibited the migration of TNF-α-activated monocytes derived from the mouse spleen. When this M5 antibody was examined using a mouse calvaria organ culture system, bone destruction suppressive action was observed. Furthermore, when the above-described antibody was administered to a mouse model of collagen arthritis, a distinct therapeutic effect was confirmed (patent document 1 and non-patent document 5).
These results strongly suggest that an antibody that simultaneously blocks the binding between the RGD sequence and human type integrin, and between the SVVYGLR sequence (SEQ ID NO:10) and human type integrin inhibits the binding between OPN and integrin and is effective in the treatment of rheumatoid arthritis and the like, and furthermore show that the antibody is expected to be effective not only in the treatment of forms of rheumatism such as juvenile rheumatoid arthritis and chronic rheumatism, but also in the treatment of psoriatic arthritis and psoriasis. Chronic graft rejection after organ transplantation is characterized by obstructive lesions in blood vessels and bronchia; from histological investigations thereof, it is considered that because activation of T cells and macrophages causes production of cytokines and growth factors and vascular endothelial cell disorder, and also because vascular smooth muscle growth causes fibrosis and the like, the condition progresses to vascular obstruction (non-patent documents 6 to 8).
It has been reported that OPN functions as an essential protein in these macrophage activation and vascular smooth muscle fibrosis (non-patent document 9); an OPN inhibitory antibody may suppress the process toward fibrosis by suppressing the migration of monocytes and neutrophils. Therefore, the antibody is expected to suppress chronic graft rejection after organ transplantation to contribute to the take of organs, and to be effective in the treatment of autoimmune diseases such as systemic autoimmune disease, erythematosus, uveitis, Behcet disease, multiple myositis, glomeruloproliferative nephritis, and sarcoidosis. It has also been confirmed that the expression level of OPN increases in various cancers, and that OPN promotes the cancer progression and metastasis (non-patent documents 10 to 12), and that cancer cell growth and metastasis are suppressed by an anti-OPN antibody (patent document 3, non-patent document 13). Therefore, an anti-OPN antibody is also expected to be effective in the treatment of various cancers.
Disclosed in WO03/027151 (patent document 2) are a chimeric anti-human osteopontin antibody having both the variable region of the mouse anti-human osteopontin antibody 2K1 described in patent document 1 and the constant region of a human antibody, and a humanized anti-human osteopontin antibody having both the complementarity determining region of the 2K1 antibody and the framework region and constant region of a human antibody.
Meanwhile, a large number of monoclonal antibodies for treatment are already available in the market, including antibodies for cancer treatment (for example, rituximab, trastuzumab, bevacizumab), antibodies for rheumatism treatment (for example, infliximab, adalimumab), antibodies for treatment for suppressing graft rejection (for example, muromonab, basiliximab) and the like.
Because of their basic features of high specificity and safety, it seems that research and development of monoclonal antibody preparations, particularly targeting a wide variety of diseases for which low-molecular therapeutic drugs are difficult to develop, will be accelerated.
On the other hand, the greatest problem posed in the development of such antibody pharmaceuticals concerns antibody productivity. The clinical doses of monoclonal antibodies that have been launched in the market are generally on the order of several mg/kg, so that considerable production costs are required.
For this reason, to select an antibody that exhibits excellent activity and, out of antibodies showing the same activity, an antibody of high expression levels and high stability for a protein, is a very important requirement for actual application as an antibody pharmaceutical.
Patent document 1: Pamphlet for International Patent Publication No. WO02/081522
Patent document 2: Pamphlet for International Patent Publication No. WO03/027151
Patent document 3: Pamphlet for International Patent Publication No. WO06/043954
Non-patent document 1: Y. Saitoh et al., (1995): Laboratory Investigation, 72, 55-63
Non-patent document 2: Y. Yokosaki et al., (1999): The Journal of Biological Chemistry 274, 36328-36334
Non-patent document 3: P. M. Green et al., (2001): FEBS Letters 503, 75-79
Non-patent document 4: S. T. Barry et al., (2000): Experimental Cell Research 258, 342-351
Non-patent document 5: Yamamoto et al., (2003): The Journal of Clinical Investigation, 112, 181-188
Non-patent document 6: P. Freese et al., (2001): Nephrology, dialysis, transplantation, 16, 2401-2406
Non-patent document 7: J. R. Waller et al., (2001): British Journal of Surgery, 88, 1429-1441
Non-patent document 8: S. R. Lehtonen et al., (2001): Transplantation, 72, 1138-1144
Non-patent document 9: A. O'Regan et al., (2000): International Journal of Experimental Pathology, 81, 373-390
Non-patent document 10: G. F. Weber, (2001): Biochimica et Biophysica Acta, 1552, 61-85
Non-patent document 11: H. Rangaswami et al., (2006): TRENDS in Cell Biology 16, 79-87
Non-patent document 12: S. S. Forootan et al., (2006): Int. J. Cancer: 118, 2255-2261
Non-patent document 13: Z. Hu et al., (2005): Clin. Cancer Res. 11 4646-4652